Determination of endogenous levels of cyclic ADP-ribose in rat tissues

Cyclic ADP-ribose (cADPR) is a potent mediator of calcium mobilization in sea urchin eggs. The cADPR synthesizing enzyme is present not only in the eggs but also in various mammalian tissue extracts. The purpose of this study was to ascertain whether cADPR is a naturally occurring nucleotide in mammalian tissues. Rat tissues were frozen and powdered in liquid N₂, followed by extraction with perchloric acid at -10°C. [³²P]cADPR was prepared and used as a tracer. The acid extracts were chromatographed on a Mono-Q column and cADPR in the fractions were determined by its ability to release Ca²⁺ from egg homogenates. That the release was mediated by cADPR and not inositol triphosphate (IP3) in the extracts was shown by the fact that the homogenates, subsequent to Ca²⁺ release induced by active fractions, were desensitized to authentic cADPR but not to IP3. Furthermore, the Ca²⁺ release activity was shown to co-elute with [³²P]cADPR. The endogenous level of cADPR determined in rat liver is 3.37 ± 0.64 pmol/mg, in heart is 1.04 ± 0.08 pmol/mg and in brain is 2.75 ± 0.35 pmol/mg. These results indicate cADPR is a naturally occurring nucleotide and suggest that it may be a general second messenger for mobilizing intracellular Ca²⁺.